Keratinocytes useful for the treatment of wounds

ABSTRACT

The invention relates to new keratinocytes which may be cultured in vitro and the advantageous use thereof for preparing a product which can be used to treat acute and chronic wounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 10/268,496 filedOct. 10, 2002 now U.S. Pat. No. 7,247,478.

FIELD OF THE INVENTION

The invention relates to new keratinocytes which may be cultivated invitro and their beneficial use for preparing a product which can be usedto treat acute and chronic wounds. The invention was thus made in thefield of medicine, specifically in the field of wound healing by tissueengineering.

PRIOR ART

Allogeneic keratinocytes have successfully been used for quite a longtime for the treatment of wounds, particularly ulcers and/or burns(Maier, 1993; Schönfeld et al., 1993). Wounds which prove resistant totreatment by conventional methods over lengthy periods can often besuccessfully treated using allogeneic keratinocytes (Phillips andGilchrest, 1993; Beele et al., 1991; Leigh et al., 1991; Lindgren et al,1998). To begin with, when using allogeneic keratinocytes, attention wasdirected predominantly to skin replacement, i.e. the regeneration of thetransplanted epidermis. However, it was soon apparent that the successof the treatment is primarily based on the stimulation of the body's ownre-epithelialisation and not on “growing” the allogeneic cell transplantin the wound. Recently, numerous investigations have shown that thetransplanted keratinocytes remain in the wound for only a certain lengthof time and are then eliminated from the body in a manner which isclinically invisible (Kawai et al., 1993). The stimulation of the body'sown wound healing is thus primarily brought about by an optimum release,in space and time, of a plurality of different growth factors,cytokines, extracellular matrix (ECM), smaller molecules and proteasesby the allogeneic keratinocytes (Lang et al., 1996; Marcusson et al.,1992). The complexity and number of factors released confirms thetherapeutic advantage over conventional single therapies, e.g. withisolated growth factors. Apart from the main effect ofre-epithelialisation, however, treatment with keratinocytes alsoproduces macroscopically and microscopically detectable changes in thegranulation tissue (Lang et al., 1996). Another frequently describedeffect of keratinocytes which is beneficial to the patient is the markedanalgesia after transplantation (Schönfeld et al., 1993).

For wound healing it is advantageous to use undifferentiated, activelydividing keratinocytes, as actively dividing keratinocytes appear tohave a complex secretion profile which promotes wound healing. With theexception of the stem cells, undifferentiated keratinocytes lose theirproliferation potential in vivo in the course of natural differentiationafter only a few cell divisions, a process which has hitherto alwaysbeen observed in the in vitro cultivation of keratinocytes (Barrandonand Green, 1987). Consequently, the actively dividing keratinocyteswhich are particularly suitable for wound healing have hitherto onlybeen cultivated in vitro, which makes them difficult and expensive touse for medical purposes.

The allogeneic keratinocytes are used therapeutically either directly inthe form of so-called “keratinocyte sheets”, consisting of a multilayer,enzymatically detached cell aggregate, or together with biocompatiblecarriers under the title “biologically active wound healing dressings”.The latter has the advantage that the cells do not require enzymaticpretreatment, detaching them from the substrate of the culture dishbefore they are used. Moreover, the use of biocompatible membranes ascarriers for cultivating the keratinocytes (EP 0 462 462, U.S. Pat. No.5,658,331; U.S. Pat. No. 5,693,332; EP 0518 920) makes it possible toprepare the biologically active wound healing dressings earlier, as thecells no longer have to form a self-contained cell aggregate. Carrierswhich have already grown subconfluent may be used for the woundtreatment. Apart from being available sooner, the use of subconfluentcell aggregates has the additional advantage that correspondinglycultured keratinocytes are less sharply differentiated and are thusadvantageous for treating wounds.

Keratinocytes may be cryopreserved for wound healing, without anyharmful effects, directly or in conjunction with biocompatible carriermaterials at temperatures between −30 and −196° C., but preferablybetween −70 and −90° C. (De Luca et al., 1992; Teepe et al., 1993). Thisfurther improves their therapeutic usefulness, as it is possible to someextent to keep a supply of biologically active wound healing dressings.

PROBLEM OF THE INVENTION

The keratinocytes known from the prior art and biologically active woundhealing dressings produced from them have certain drawbacks which thepresent invention set out to overcome.

An obvious disadvantage of the keratinocytes of primary origin knownhitherto is that the said cells can only replicate themselves for a fewgenerations of cells by means of in vitro cell culture processes withoutlosing their high rate of division and hence their suitability for thepreparation of biologically active wound healing dressings. According tothe prior art the skilled man is only able to increase the number ofcells by a factor of about 10³ to 10⁴ by in vitro cultivation (Tanczoset al., 1999).

Another disadvantage which flows from this is the fact that the limitedculturability of said keratinocytes in vitro makes it essential to carryout frequent and complex re-isolation; this in turn means that thereplicated cell material obtained is not uniform and hence the woundhealing dressings produced therefrom will, or at least may, in allprobability have differences in quality.

A further disadvantage is that the re-isolation of the keratinocytesfrom various donors constitutes an increased risk of infection to therecipient of the wound healing aggregate. An increased risk of HIV orhepatitis might arise, for example.

DESCRIPTION OF THE INVENTION

The invention relates to keratinocytes with a high proliferationpotential, which are not immortalised and which can be replicated atleast 150 times by in vitro cell culture methods. This results in a cellmultiplication factor of about 10⁴⁴. The corresponding keratinocytesstill retain their advantageous properties for the treatment of wounds.

The keratinocytes according to the invention are primarily keratinocytesisolated from a donor and culturable in vitro, while the isolation andinitial cultivation may be carried out by anyone skilled in the art,according to the process described by Rheinwald and Green in 1975, forexample.

The invention preferably relates to keratinocytes which are isolatedfrom the epidermal part of a foreskin. Keratinocytes of human origin,particularly keratinocytes of the culture KC-BI-1, which were depositedon 27 Jun. 2001 at the DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen [German Collection of Microorganisms and Cell Cultures]GmbH, Braunschweig, Germany under Accession Number DSM ACC2514 for thepurposes of patent proceedings according to the Budapest Agreement, arepreferred. The invention also covers keratinocytes which are derivedfrom the culture KC-BI-1 (DSM ACC2514). Thus, the invention also relatesto all cells and cultures which are and/or may be generated bysubpassaging and/or subcloning the original culture KC-BI-1.

The cultivation of the keratinocytes according to the invention isdescribed by way of example in relation to the keratinocytes KC-BI-1(Example 1). The use of the complex medium specified in Example 1 andthe use of feeder cells, preferably the use of lethally irradiatedmurine 3T3 fibroblasts, is advantageous for the culturing. The amount offoetal calf serum should be between 2 and 10%. The preparation of thefeeder cells is known to those skilled in the art and may be carried outfor example by the process described in Example 2. Subcultivation of thekeratinocytes according to the invention at a maximum confluence of 80%is particularly advantageous. The keratinocytes may be cultured at 35 to38° C., preferably at 37° C., at a relative humidity of >90%, preferably95% and a CO₂ saturation of 5 to 9%.

With the process described in Example 1 the population doubling time forthe keratinocytes, particularly human keratinocytes from the epidermalpart of a foreskin, is between 1 and 2 days (FIG. 1). The cells can becultured over numerous passages at a substantially constant replicationrate (FIG. 2). However, the present invention is not restricted to justthe keratinocytes KC-BI-1, but rather it is possible for a skilled manto perform the invention under appropriate conditions with anykeratinocytes that are not immortalized and may be doubled at least 150times by in vitro cell culture methods.

The expression “not immortalised” in relation to the present inventionmeans that primarily isolated keratinocytes and the keratinocytescultured here are not spontaneously transformed and/or have not beentransformed by molecular-biological, chemical or physical methods knownfrom the research. The latter means that the cells have not been treatedeither using e.g. viral factors or sequences, chemically mutagenicsubstances or, for example, by irradiation or a combination of differentprocesses.

Keratinocytes which “are not immortalised” also means that compared withtransformed tumour cell lines (Härle-Bachor and Boukamp, 1993) orcompared with immortalised cell lines, preferably the cell line HeLa,the said keratinocytes have no telomerase activity or a substantiallyreduced telomerase activity (cf. FIG. 3). This is particularly true alsoin comparison with the immortalised keratinocyte cell line HaCat.

The expression “not immortalised” also means that said keratinocytescannot be replicated in the absence of foetal calf serum and/or in theabsence of feeder cells and/or in the absence of epidermal growthfactor, EGF, as immortalised keratinocytes can, for example (Schoop etal., 1999).

The expression “not immortalised” also means, however, that saidkeratinocytes do not change their characteristic phenotype as the cellreplication increases (cf. FIG. 4).

“Not immortalised” also means that the said keratinocytes exhibit anormal differentiation profile after transplantation onto nude mice,preferably BALB/c. “Normal differentiation potential” here means theability of the keratinocytes to develop into terminally differentiatedkeratinocytes and form suprabasal epidermal layers as well as a stratumcorneum in the same way as autologous keratinocytes.

The present invention also relates to keratinocytes which are notimmortalised and which can be replicated at least 200 times by in vitrocell culture methods. The invention further relates to keratinocyteswhich are not immortalised and can be replicated at least 250 times byin vitro cell culture methods. The invention also relates tokeratinocytes which are not immortalised and can be replicated at least300 times by in vitro cell culture methods.

The present invention advantageously enables the said keratinocytes tobe replicated from only one donor or only a few donors. Starting fromone donation, for example, 10⁴⁴ cells may be produced after 150 cellreplications, 10⁷⁷ cells after 250 cell replications and 10⁹⁰ cellsafter 300 cell replications. Thus, the invention makes it possible forthe first time to produce large quantities of standardised cell materialfor the preparation of biologically active wound healing aggregates ofconstant, verifiable quality. A corresponding amount of standardisedcell material may be replicated, for example, starting from acryopreserved cell bank, which is in turn produced from keratinocyteshaving the properties according to the invention.

By using standardised cell material as the starting material forpreparing biologically active wound healing dressings the risk ofinfection to the possible recipients is also reduced, as the isolationof the keratinocytes is restricted to a few donors, preferably onedonor.

Thus, the invention overcomes serious disadvantages which currentlyconsist of the merely restricted passaging of the keratinocytes knownhitherto.

The present invention further relates to a product which consists of acarrier coated with the keratinocytes according to the invention.“Coated” for the purposes of the invention means that the surface of thecarrier is partially or totally colonised with the keratinocytesaccording to the invention. A partially colonised carrier isparticularly suitable, as a shorter culture time is needed before thecarrier can be used for wound treatment.

A suitable carrier for the purposes of the invention is characterised inthat it is a biocompatible carrier material which may be used to preparea pharmaceutical composition. Hydrophobic biocompatible carriermaterials, as described in WO 91/13638, for example, may be used.However, it is also possible to use carrier materials with predominantlyhydrophilic properties.

A preferred embodiment of the present invention comprises the use ofcarrier materials which consist of a polymer of esterified hyaluronicacid. In a particularly preferred embodiment a polymer of esterifiedhyaluronic acid is used, consisting of a perforated polymer film of adefined geometry. The polymer film has a thickness of 10 to 500 μm, forexample, and is perforated with holes measuring between 10 and 1000 μm,the holes being of a defined, constant size and forming ordered rows,separated from one another by a constant spacing of 50 to 1000 μm. Afilm of this kind is described in EP 0 462 426. Perforated carriermaterials are particularly suitable as they do not require thebiologically active wound dressing to be placed on the wound in anyparticular direction. Example 3 describes the preparation, by way ofexample, of a perforated carrier matrix of esterified hyaluronic acid ofa defined geometry colonised by keratinocytes according to theinvention. The carrier matrix is a product made by Messrs Fidia AdvancedBiopolymers Ltd., Abano Terme, Italy, marketed in Germany under theproduct name “Laserskin”.

The particularly preferred suitability of this carrier material forproducing a biologically active wound dressing in conjunction withkeratinocytes has already been demonstrated on an animal model (Lam etal., 1999) and in humans (Harris et al., 1999). Apart from improvedmigration and differentiation of the epithelial cells the matrixconsisting of hyaluronic acid ester has a positive effect onangiogenesis and collagen production. The wound dressings currently usedwith hyaluronic acid ester as matrix are, however, coated withautologous keratinocytes and/or skin equivalents of complex structureobtained from keratinocytes and fibroblasts. They therefore suffer fromthe disadvantages of the prior art described above. These disadvantagesmay be overcome particularly by the use of the advantageous allogeneickeratinocytes according to the invention.

Another preferred embodiment of the invention relates to a productcomprising the keratinocytes according to the invention together withreabsorbable polymers, consisting of polyesters, polycarbonates,polyanhydrides, polyorthoesters, polydepsipeptides, polyetheresters,polyamino acids or polyphosphazenes, especially poly(L-lactide),poly(D,L-lactide), poly(L-lactide-co-D,L-lactide), poly(glycolide),poly(L-lactide-co-glycolide), poly(L-lactide-co-trimethylene-carbonate)or poly(dioxanone). These polymers are both perforated and unperforated.

The present invention also relates to a method of cryopreserving thekeratinocytes according to the invention at a temperature of −20° C. to−196° C., preferably at a temperature below −180° C. The saidkeratinocytes can be frozen by standardised methods familiar to anyoneskilled in the art. DMSO, inter alia, may be used as the cryoprotectant.It is also possible to use other cryoprotectants such as glycerol,hydroxyethyl starch or a combination of the two, and a combination ofthese with DMSO. Suitable methods are described for example in WO96/24018, U.S. Pat. No. 5,891,617 or U.S. Pat. No. 5,298,417.

The present invention also relates to the cryopreserving of the carrierscoated with the keratinocytes according to the invention, characterisedin that the keratinocytes with their corresponding carrier arecryopreserved at a temperature of −20° C. to −196° C., preferably at−60° C. to −80° C. The advantage of cryopreserving is that the productobtained in large quantities can be stored and thus examined foruniformity of quality by random sampling before clinical use. Finally,storage ensures that the wound healing aggregates are available at shortnotice for medical purposes.

A suitable cryoprotectant for the product according to the invention ishydroxyethyl starch, for example, in a concentration of 7-13% (w/w).However, it is also possible to use DMSO or glycerol as well as acombination of various cryoprotectants, particularly hydroxyethylstarch, DMSO and/or glycerol. It is also possible to use trehalose ascryoprotectant.

After a rapid reduction in temperature from 37° C. to −5 to −10° C.,preferably −6 to −8° C. within 2-5 min, the product comprising carrierand keratinocytes according to the invention is equilibrated at theappropriate temperature for 15-30 min, preferably for 23-26 min. Thenthe product is cooled at a freezing rate of <1° C./min, preferably from0.2 to 0.6° C./min, most preferably 0.4° C./min, to a temperature ofe.g. −60 to −80° C.

Example 4 describes, by way of example, the cryopreserving of a carriermatrix of hyaluronic acid ester coated with KC-BI-1. However, it is alsopossible to use other methods of cryopreserving, e.g. the methodsdescribed in WO 95/707611, WO 96/24018, EP 0 296 475; this list shouldnot be regarded as exhaustive but merely indicates that methods ofcryopreserving products consisting of biocompatible carriers andkeratinocytes are part of the current state of the art.

The present invention also relates to the medical use of thekeratinocytes according to the invention described here and/or theproduct of said keratinocytes and a carrier described here, particularlyto their use for treating wounds. One embodiment of the inventionconsists of the use of the keratinocytes according to the inventionand/or the product of said keratinocytes and a carrier in the treatmentof burns and/or ulcers. Burns which may be treated are preferably seconddegree burns while the ulcers are preferably chronic ulcers of the lowerleg which are difficult to heal, of the type Ulcus cruris, preferablyUlcus cruris venosum or diabetic ulcers, and also decubital ulcers.

The medical use includes a combined and/or supplementary use of saidkeratinocytes and/or the product consisting of keratinocytes and carrieraccording to the invention with conventional therapies known in the artwith a beneficial effect on wound healing. This means a combined and/orsupplementary use of one or more other substance(s) with a beneficialeffect on wound healing. Mention may be made of the supplementary and/orcombined treatment of Ulcus cruris venosum with hydrocolloid dressingsand/or the additional use of antimicrobial substances, e.g. theadministering of antibiotics.

The invention also relates to the keratinocytes according to theinvention and/or the product of a biocompatible carrier and saidkeratinocytes for preparing a medical product for treating wounds,particularly for treating burns and/or ulcers, e.g. for the treatment ofsecond degree burns, Ulcus cruris (venosum), diabetic ulcers ordecubital ulcers.

The invention further relates to a process for treating these wounds,this process being characterised in that the keratinocytes according tothe invention and/or the product according to the invention comprisingkeratinocytes and carrier is or are placed on the wounds to be treated.The keratinocytes and the product may be used either fresh or aftercryopreservation. A corresponding method of treating wounds is describedin Example 5.

DESCRIPTION OF THE FIGURES

FIG. 1: Cell replication of the keratinocytes KC-BI-1 as a function ofthe culture time. This shows the number of cell replications(CPD=Cumulative Population Doublings) of the keratinocytes KC-BI-1 overa culture period of 94 days. Within the 94 day observation period thecells doubled roughly 75 times. This corresponds to a mean doubling timeof 1.25 days per cell replication, or 12.5 per 10 days.

FIG. 2: Doubling of the keratinocytes KC-BI-1 over a period of 10months. This shows the cell replication of the keratinocytes KC-BI-1over 10 months, given as population doublings (PD) as a function of thecell passages 1-67.

FIG. 3: Determining the relative telomerase activity. This shows therelative telomerase activity for the keratinocytes KC-BI-1 after passage1, 12, 18, 40 and 57 compared with the activity of the cell line HeLa.The Figure shows almost no or only slight telomerase activity for thekeratinocytes KC-BI-1 compared with the immortalised cell line HeLa.

FIG. 4: Morphology of the keratinocytes KC-BI-1 after passages 5 and 60.Viewed under the optical microscope the keratinocytes KC-BI-1 do notexhibit any morphological differences between cell passage 5 (culturetime 25 days) and cell passage 60 (culture time 300 days).

EXEMPLIFYING EMBODIMENTS Example 1 Process for Culturing theKeratinocytes According to the Invention Taking the Culture KC-BI-1 (DSMACC2514) as an Example

1. Material

Keratinocytes KC-BI-1; irradiated 3T3-murine fibroblasts (feeder cells,for preparation cf. Example 2); cell culture medium K/1 (for compositionsee below); EDTA (0.02%); trypsin/EDTA (0.05%/0.01%); cell cultureflasks (T-flask): 25 cm², 80 cm², 175 cm²

2. Thawing the Cells

2.1 Thawing the Feeder Cells

The cells are rapidly thawed and placed in 5-10 ml of preheated K/1medium. A corresponding quantity of feeder cells are transferred into asuitable cell culture flask and topped up with K/1 medium:

 25 cm² T-flask 0.5 × 10⁶ cells Final volume of medium: 5-6 ml  80 cm²T-flask 1.5 × 10⁶ cells Final volume of medium: 20 ml 175 cm² T-flask3.5 × 10⁶ cells Final volume of medium: 50 ml

The feeder cells may be used immediately or within 24 hours.

2.2 Thawing the Keratinocytes

The cells are rapidly thawed and placed in 5-10 ml of preheated K/1medium. A corresponding quantity of cells are added to the cell cultureflasks already containing feeder cells and topped up with fresh medium:

 25 cm² T-flask 0.15 × 10⁶ cells; Final volume of medium: 6-10 mL  80cm² T-flask  0.4 × 10⁶ cells; Final volume of medium: 20 mL 175 cm²T-flask   1 × 10⁶ cells; Final volume of medium: 50 mL3.0 Cultivation

The cells are incubated at 35-39° C., preferably at 37° C. The relativehumidity is >90%, preferably 95% and the CO₂ concentration is 5-9%. Thekeratinocytes are subcultured at a maximum confluence of 80%.

For this, the cell culture supernatant is discarded. The feeder cellsare rinsed twice with 0.02% EDTA (2-10 ml) and incubated for 5-10 min at37° C., then detached from the cell culture flask by tapping it (orshaking it). The keratinocytes are then treated with trypsin/EDTA(0.05%/0.01%, 1-6 ml) for 5-10 min at 37° C. and carefully detached bytapping. If necessary the remaining cells are carefully scraped offusing a cell spatula. The trypsin/EDTA solution is neutralised by theaddition of K/1 medium and the cells are separated by careful pipettingup and down. The cells are seeded out in the cell numbers specified in2.2. The cell culture medium K/1 is changed on day 3 and then every twodays.

4.0 Preparation of the K/1 Medium

All the components and the stock solutions are combined one afteranother in the sequence given in Table 1. The mixture is made up to 1litre with WFI (water for injection). Then the pH is adjusted to 7.0 to7.2 with NaOH or HCl. The osmolarity should be between 320-400 mOsm/kg.Finally, the medium K/1 is sterile-filtered.

4.1 Preparation of the Stock Solutions

Triiodothyronine

13.6 mg of triiodothyronine are dissolved in 1 ml of 0.1 NaOH and 99 mlof PBS are added thereto. The finished solution is diluted 1:100 in PBS.1 ml of this solution is required per litre of medium.

EGF

1 mg of EGF is dissolved in 100 ml of WFI. 1 ml of this solution isrequired per litre of medium.

rh-Insulin (Only Soluble at pH <4.0)

5 g of rh-insulin are added to 0.9 L of WFI and the pH is adjusted to2.5 with 6M HCl. After the rh-insulin has dissolved, the pH is adjustedto 8.0 with 1M NaOH. The mixture is made up to 1 litre with WFI. 1 ml ofthis solution is required per litre of medium.

4.2 Composition of the Medium

components concentration/L WFI (water for injection) 0.8 L DMEM withglutamine 10.035 g HAM's F12 2.66 g sodium hydrogen carbonate 3.07 gNa-pyruvate 0.11 g Apo-Transferrin 5 mg Adenine-Hemisulphate 147.4 mgForskolin (soluble in a few drops of DMSO) 2.05 mgRh-Insulin-Concentrate 1.0 mL Hydrocortisone 10 mM 0.11 mLtriiodothyronine stock solution 1.0 mL EGF-stock solution 1.0 mL Phenolred 8.1 mg FCS (2-10%) 20-100 mL 6 M HCl as required 40% NaOH asrequired WFI add to 1.0 L

However, the cells may also be cultured from fresh biopsy material, e.g.from the epidermal part of a foreskin. The primary isolation of theundifferentiated, proliferating keratinocytes may be carried out usingthe method described by Rheinwald and Green in 1975.

The feeder cells used may be, for example, the cells described inExample 2. It is also conceivable to use other lethal fibroblasts,preferably other murine fibroblasts, most preferably descendants of cellline 3T3.

Example 2 Preparation of Irradiated 3T3 Feeder Cells for CultivatingKeratinocytes

1. Material

Murine 3T3 fibroblasts (e.g. ATCC CCL 92, 3T3-Swiss albino,contact-inhibited fibroblasts) which may be obtained from the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va., USA, DMEM+10% foetal calf serum (FCS); PBS; 0.2% trypsin solution;0.04% EDTA solution; cell culture flasks (T-flasks): 25 cm², 80 cm², 175cm²

2. Thawing the Cells

The cells are rapidly thawed and added to 5-10 ml of preheated medium.DMSO-containing medium is removed after centrifugation. The cells aresuspended in 5-10 ml of medium. After the cell number has beendetermined the cells are seeded into suitable cell culture flasks in adensity of 10³ to 10⁴ cells/cm². They are incubated at 35-39° C.,preferably at 37° C. The relative humidity is >90%, preferably 95% andthe CO₂ concentration is 5-9%.

3. Culturing the Cells

The cells are inspected daily for growth. The cell density should notexceed a maximum confluence of 70-80%. Subculturing is carried out, asnecessary, every 2 to 4 days. For this, the medium is discarded and thecells are washed with a suitable amount of a 1:2 mixture of EDTA andtrypsin (0.5-5 ml). Then the cells detached are taken up in 3.5-20 ml ofmedium. The cells are re-seeded at a density of 10³ to 10⁴ cells/cm².

4. Irradiation of the Feeder Cells

The feeder cells are irradiated with a dose of about 60 Gy (6000 rad ina ¹³⁷Cs source).

Both the irradiated and the non-irradiated cells can be cryopreserved inliquid nitrogen using standard methods and stored for long periods.

Example 3 Method of Coating a Carrier Matrix, in this Case Laserskin,with Keratinocytes from the Culture KC-BI-1 (DSM ACC2514)

The preparation of the biologically active wound healing dressingaccording to the invention will now be described by way of example. Thewound healing dressing described here consists of the keratinocytesKC-BI-1 according to the invention and Laserskin, a bioreabsorbablecarrier matrix of hyaluronic acid ester.

However, the invention is not restricted to the combination describedhere. Rather, any keratinocytes which have the novel properties of notbeing immortalized and may be doubled at least 150 times by in vitrocell culture methods may be used for the coating.

It is also possible to use other suitable carrier matrices, providedthat they are biocompatible carrier materials which may be used toprepare a pharmaceutical composition. For example, hydrophobicbiocompatible carrier materials as described in WO 91/13638 may be used.In addition, however, it is also possible to use carrier materials withpredominantly hydrophilic properties.

Another preferred embodiment of the invention comprises using thekeratinocytes according to the invention together with reabsorbablepolymers, consisting of polyesters, polycarbonates, polyanhydrides,polyorthoesters, polydepsipeptides, polyetheresters, polyamino acids orpolyphosphazenes, especially poly(L-lactide), poly(D,L-lactide),poly(L-lactide-co-D,L-lactide), poly(glycolide),poly(L-lactide-co-glycolide), poly(L-lactide-co-trimethylene-carbonate)or poly(dioxanone), and using perforated films consisting of saidpolymers.

1. Material

K/1 medium (cf. Example 1); PBS, 0.04% EDTA (diluted to 0.02% with PBS);trypsin/EDTA (0.05%/0.01%); sterile Roux dishes (T 25 cm², T 80 cm², T175 cm²,), Laserskin (Messrs. Fidia Advanced Biopolymers srl, AbanoTerme, Italy) in 144×21 Petri dishes (145 cm² surface area); 3T3 feedercells; keratinocytes according to the invention such as KC-BI-1 forexample

2. Culturing the Biologically Active Wound Healing Dressing

2.1. Material

Irradiated feeder cells, e.g. the murine 3T3 fibroblasts mentioned inExample 2; keratinocytes according to the invention from stock; 8.5cm×8.5 cm pieces of Laserskin in a Petri dish (finished product); K/2medium

2.2. Seeding Out the 3T3 Feeder Cells

The feeder cells, prepared according to Example 2, are placed onLaserskin in a seeding density of about 15,000 to 25,000 cells/cm²(roughly corresponding to 3×10⁶ cells/Petri dish). The Petri dish isthen incubated at 35 to 37° C. at >90% relative humidity and 5-11% CO₂,preferably 7-9%, in an incubator at 37° C. The keratinocytes are seededonto the feeder cell lawn either on the same day or, at the latest, thenext day (after 24 hours).

2.3. Seeding Out and Culturing the Keratinocytes

The biologically active wound healing dressings are prepared with thekeratinocytes according to the invention. The subculturing of thekeratinocytes may, for example, be carried out as follows:

The subconfluent cultures are rinsed once with 0.02% EDTA (80 cm² Rouxdish: 8 mL; 175 cm² Roux dish: 10 ml). Then the feeder cells areincubated with 0.02% EDTA for 5-10 min at 37° C. (80 cm² Roux dish: 8mL; 175 cm² Roux dish: 10 ml) and detached by shaking carefully.

The keratinocytes are dissolved as in Example 1 with a trypsin/EDTAmixture (0.05%/0.01%) (80 cm² Roux dish: 2-3 mL; 175 cm² Roux dish: 5-6ml), then taken up in cell culture medium (80 cm² Roux dish: 7-8 mL; 175cm² Roux dish: 14-15 ml) and separated by carefully pipetting up anddown.

The keratinocytes according to the invention are applied to theLaserskin film provided with feeder cells, in a seeding density of about15,000 to 25,000 cells/cm² (roughly corresponding to 3×10⁶ cells/Petridish). Then the cells are incubated until 30-100% confluent, preferably80-100% confluent, at 35-39° C., preferably at 37° C. The relativehumidity is >90%, preferably 95% and the CO₂ concentration is 5-9%.

Example 4 Method of Cryopreserving the Biologically Active Wound HealingDressing According to the Invention

After the keratinocytes have colonised the carrier matrix to aconfluence of 30-100%, preferably 80-100%, the product according to theinvention may be frozen in suitable containers, e.g. heat-sealable PPbags, under controlled conditions. To do this, culture medium iscarefully removed and replaced by 20 ml of K/2 freezing medium at atemperature of 2-6° C. The product is then packaged under sterileconditions and frozen according to the following procedure:

After a rapid lowering of the temperature to −5 to −10° C., preferably−6 to −8° C. within 2-5 min, the product is equilibrated at thecorresponding temperature for 15-30 min, preferably for 23-25 min. Thenthe product is cooled to a temperature of, for example, −60 to −80° C.at a freezing rate of <1° C./min, preferably 0.2 to 0.6° C./min, mostpreferably 0.4° C./min. The product is stored at −60 to −80° C.

K/2 Freezing Medium:

K/1 growth medium (cf. Example 1) mixed with 7-13% (w/w) of hydroxyethylstarch.

Example 5 Example of the Use of a Carrier Matrix Colonised with theKeratinocytes According to the Invention for Covering Wounds, TakingVenous Leg Ulcers as an Example

1. Transporting Freshly Prepared Wound Healing Dressings

After the keratinocytes have grown to 30-100%, preferably 80-100%confluence on the Laserskin, the culture is rinsed one or more timeswith a suitable quantity, preferably 30 ml, of K/3 transporting medium.The biologically active wound healing dressing is transported in asuitable amount, preferably in 20 ml, of K/3 transporting medium. Theheadroom of the Petri dish is briefly gassed with an air mixture of5-10% CO₂, sealed with adhesive tape, e.g. Parafilm, and immediatelydelivered to the clinic in a transportation box.

K/3 Transporting Medium:

Growth medium K/1 (cf. Example 1) without foetal calf serum (FCS).However, it is also possible to use simple physiological salinesolutions e.g. based on phosphate-borate, e.g. PBS, or based on HEPES(N-2-hydroxyethylpiperazin-N′-2-ethanesulphonic acid) or MES([2-N-morpholino] ethanesulphonic acid).

2. Transporting Cryopreserved Wound Healing Dressings

Cryopreserved wound healing dressings may typically be supplied to theclinics on dry ice. However, other forms of transportation are possible,provided that the wound healing dressings are transported at atemperature below −60° C. The cryopreserved wound healing dressings arerapidly thawed. Then the freezing medium is removed and the dressing isrinsed one or more times with K/3 transporting medium (see above) oranother suitable physiological solution such as Ringer's solution, forexample.

3. Therapeutic Use

The dressing is then placed on the wound. When non-perforated carriermaterials are used, the wound healing dressing has to be positionedcorrectly with the cells facing the wound. The use of perforatedcarriers which allow keratinocytes to colonise both sides of the carrier(e.g. Laserskin) means that the wound healing dressing according to theinvention does not have to be placed on the wound being treated in anyparticular direction. Depending on the success of the therapy thetreatment may be repeated a number of times.

Example 6 Genetic Characterisation of the Keratinocyte Cell KC-BI-1 (DSMACC2514)

KC-BI-1 cells were subcultured over a number of passages using themethod according to the invention described above. Cells from passage 4,13 and 121 were then subjected to genetic analysis, investigating thelength polymorphism of 15 different loci (CSF1PO, D13S317, D16S539,D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA, Penta D, Penta E,TH01, TPOX and vWA). Analysis was carried out using a method known inthe art. For this, the corresponding alleles were amplified using a testto determine paternity (PoewerPlex® 16 System) produced by MessrsPromega (Mannheim, Germany), according to the manufacturer'sinstructions. The alleles may be identified by determining the fragmentlength (length standard ILS 600 is part of the above kit). Data onallele frequencies in the population can be found in the correspondingTables.

Analysis has shown agreement of all the alleles at all the loci for allthe cell passages analysed. The data thus enable the KC-BI-1 cells to begenetically classified. A classification probability of >99.999% wasdetermined from the allele frequencies.

Determining the DNA Length Polymorphism:

KC-BI-1 KC-BI-1 KC-BI-1 Marker Donor Passage 4 Passage 13 Passage 121CSF1PO 11 11 11 11 12 12 12 12 D13S317 8 8 8 8 13 13 13 13 D16S539 11 1111 11 13 13 13 13 D18S51 13 13 13 13 17 17 17 17 D21S11 29 29 29 29 2929 29 29 D3S1358 15 15 15 15 15 15 15 15 D5S818 9 9 9 9 11 11 11 11D7S820 9 9 9 9 9 9 9 9 D8S1179 10 10 10 10 14 14 14 14 FGA 23 23 23 2326 26 26 26 Penta D 11 11 11 11 12 12 12 12 Penta E 10 10 10 10 12 12 1212 TH01 8 8 8 8 9.3 9.3 9.3 9.3 TPOX 8 8 8 8 10 10 10 10 vWA 16 16 16 1618 18 18 18

LITERATURE

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1. A method for treating a wound of the skin which comprises applying tothe wound a preparation of isolated human keratinocytes that are notimmortalised and that may be replicated at least 150 times by in vitrocell culture methods, which keratinocytes are cells from the cultureKC-BI-1 (DSM ACC 2514) or keratinocytes derived therefrom.
 2. A methodof treating a wound of the skin according to claim 1 wherein thepreparation further comprises a carrier onto which the keratinocytes arecoated and wherein the carrier is at least partially colonized with saidkeratinocytes.
 3. The method of claim 1 or 2, wherein the wound is a bumor ulcer.
 4. The method of claim 1 or 2, wherein the wound is a seconddegree bum.
 5. The method of claim 1 or 2, wherein the wound is a lowerleg ulcer of the type Ulcus cruris.
 6. The method of claim 1 or 2,wherein the wound is an ulcer caused by diabetes.
 7. The method of claim1 or 2, wherein the wound is a decubital ulcer.